Vanadate, an inhibitor of growth, development and endocytosis in Dictyostelium discoideum amoebae

1989 ◽  
Vol 94 (1) ◽  
pp. 127-134
Author(s):  
G.É. KLEIN ◽  
DAVID A. COTTER ◽  
JEAN-BAPTISTE MARTIN ◽  
MICHEL SATRE
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzymes ◽  
Fluid Phase ◽  
Nucleoside Triphosphate ◽  
Cytosolic Ph ◽  
Developmental Program ◽  
Slime Mold Dictyostelium Discoideum ◽  
Vanadate Inhibition ◽  
Massive Cell ◽  
Axenic Growth

Axenic growth of amoebae of the slime mold Dictyostelium discoideum was found to be reversibly inhibited by vanadate. Pinocytosis, when measured with fluorescein-labeled dextran as a fluorescent fluid-phase marker was strongly inhibited by vanadate. Inhibition was observable at vanadate concentrations as low as 0*2 mM. Sucrose entry through pinocytosis induced massive cell vacuolation and this effect was blocked by vanadate. Secretion of soluble lysosomal enzymes is another aspect of membrane traffic in Dictyostelium. Secretion of two typical lysosomal enzymes, acid phosphatase and hexosaminidase, was inhibited by concentrations of vanadate in the same range as for pinocytosis inhibition. Vanadate also prevented the morphogenetic developmental program that follows nutrient starvation. In contrast, vanadate did not prevent heat-induced spore germination. Vanadate had no significant action on the intracellular nucleoside triphosphate level or on the cytosolic pH. It is suggested that the particular effect of vanadate in Dictyostelium is to inhibit the fusion of endosomes with lysosomes. Our results provide a probe that could be useful to clarify the mechanisms of endocytosis.

scholarly journals Evidence for a recycling role for Rab7 in regulating a late step in endocytosis and in retention of lysosomal enzymes in Dictyostelium discoideum.

1997 ◽  
Vol 8 (7) ◽  
pp. 1343-1360 ◽  
Author(s):  
G Buczynski ◽  
J Bush ◽  
L Zhang ◽  
J Rodriguez-Paris ◽  
J Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Cell Lines ◽  
Lysosomal Enzymes ◽  
Amino Acid Level ◽  
Active Form ◽  
Fluid Phase ◽  
Retrograde Transport ◽  
Dominant Negative ◽  
Lysosomal Hydrolases ◽  
Small Molecular Weight

The mammalian small molecular weight GTPase Rab7 (Ypt7 in yeast) has been implicated in regulating membrane traffic at postinternalization steps along the endosomal pathway. A cDNA encoding a protein 85% identical at the amino acid level to mammalian Rab7 has been cloned from Dictyostelium discoideum. Subcellular fractionation and immunofluorescence microscopy indicated that Rab7 was enriched in lysosomes, postlysosomes, and maturing phagosomes. Cell lines were generated that overexposed Rab7 wild-type (WT), Rab7 Q67L (constitutively active form), and Rab7 T22N (dominant negative form) proteins. The Rab7 T22N cell line internalized fluid phase markers and latex beads (phagocytosis) at one-third the rate of control cells, whereas Rab7 WT and Rab7 Q67L cell lines were normal in uptake rates but exocytosed fluid phase faster than control cells. In contrast, fluid phase markers resided in acidic compartments for longer periods of time and were more slowly exocytosed from Rab7 T22N cells as compared with control cells. Light microscopy indicated that Rab7-expressing cell lines contained morphologically altered endosomal compartments. Compared with control cells, Rab7 WT- and Rab7 Q67L-expressing cells contained a reduced number of vesicles, the size of postlysosomes (> 2.5 microns) and an increased number of smaller vesicles, many of which were nonacidic; in control cells, > 90% of the smaller vesicles were acidic. In contrast, Rab7 T22N cells contained an increased proportion of large acidic vesicles relative to nonacidic vesicles. Radiolabel pulse-chase experiments indicated that all of the cell lines processed and targeted lysosomal alpha-mannosidase normally, indicating the lack of a significant role for Rab7 in the targeting pathway; however, retention of mature lysosomal hydrolases was affected in Rab7 WT and Rab7 T22N cell lines. Contrary to the results observed for the fluid phase efflux experiments, Rab7 T22N cells oversecreted alpha-mannosidase, whereas Rab7 WT cells retained this hydrolase as compared with control cells. These data support a model that Rab7 may regulate retrograde transport of lysosomal enzymes and the V-type H(+)-ATPase from postlysosomes to lysosomes coupled with the efficient release of fluid phase from cells.

Lysosomal enzyme secretory mutants of Dictyostelium discoideum

1990 ◽  
Vol 96 (3) ◽  
pp. 491-500
Author(s):  
D.L. Ebert ◽  
K.B. Jordan ◽  
R.L. Dimond
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzyme ◽  
High Frequency ◽  
Lysosomal Enzymes ◽  
Enzyme Modification ◽  
Regulation Of Secretion ◽  
Low Ionic Strength ◽  
Enzyme Targeting ◽  
Axenic Growth

Dictyostelium discoideum secretes a number of lysosomal enzymes during axenic growth and upon suspension in a low ionic strength, non-nutrient buffer (standard secretion conditions). These secretory characteristics have allowed us to identify 74 lysosomal enzyme secretory mutants generated by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. The majority of these mutants fell into one of four classes, on the basis of their secretory characteristics in non-nutrient buffer. The four mutant classes indicate that a minimum of three distinct sets of genes are necessary for proper secretion of lysosomal enzymes from D. discoideum cells under standard secretion conditions: one set of genes that is involved in general lysosomal enzyme secretion, one that is involved in glycosidase type secretion, and a third that is involved in acid phosphatase type secretion. These three classes likely reflect heterogeneity in the intracellular destination of lysosomal enzymes, the secretory mechanism, or both. A fourth set of genes may be necessary for proper secretion during growth, but plays no role under standard secretion conditions. These are likely altered in the regulation of secretion or in lysosomal enzyme targeting. Of the 74 secretory mutants, 36 were also modification mutants resulting in decreased pI, thermolability, or in vivo instability of lysosomal enzyme activities. The high frequency of modification mutants indicates an integral relationship between lysosomal enzyme modification, and lysosomal enzyme targeting and secretion in D. discoideum.

Dictyostelium Discoideum Mutants Resistant to the Toxic Action of Methylene Diphosphonate are Defective in Endocytosis

1992 ◽  
Vol 101 (1) ◽  
pp. 139-144 ◽  
Author(s):  
MIREILLE BOF ◽  
FRANÇOISE BRÉNOT ◽  
CARLOS GONZALEZ ◽  
GÉRARD KLEIN ◽  
JEAN-BAPTISTE MARTIN ◽  
...  
Keyword(s):  
Dictyostelium Discoideum ◽  
Lysosomal Enzymes ◽  
Fluid Phase ◽  
Screening Strategy ◽  
31P Nmr Spectroscopy ◽  
Lysosomal Hydrolases ◽  
Methylene Diphosphonate ◽  
Endosomal Pathway ◽  
Enzyme Markers

Methylene diphosphonate is taken up in Dictyostelium discoideum amoebae by fluid-phase pinocytosis, and it inhibits growth through the production of methylene analogs of adenosine triphosphate and diadenosine tetraphosphate. Methylene diphosphonate resistance was thus used as the basis of a screening strategy for the isolation of endocytosis mutants. Fifteen Dictyostelium mutants, whose growth was resistant to 7.5 mM methylene diphosphonate, were obtained and three of them were characterized in more detail. They were partially defective in fluid-phase pinocytosis (both the rate and extent of FITC-dextran entry were reduced to 40–50% of the parent type activity) and they had smaller amounts of several lysosomal enzymes, such as acid phosphatase, N-acetylhexosaminidase, α-mannosidase (20–60% of the parent type activities). In contrast to the lysosomal hydrolases, the mutants had unchanged activities for enzyme markers selective for other compartments. They appeared phenotypically similar to the Dictyostelium mutant HMW570, which is defective in fluid-phase pinocytosis and oversecretes lysosomal enzymes. The methylene diphosphonate-resistant mutants were found to be unable to acidify fully their endosomal compartments and they have an increased endosomal pH, as shown by the use of the pH-sensitive fluorescence of FITC-dextran. Furthermore, the hypothesis proposing a defective acidification of the endosomal pathway was supported by the measurement of ATP-dependent vesicular acidification with acridine orange, and by in vivo 31P NMR spectroscopy with aminomethylphosphonate as a pH probe.

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